Bottom Line:
Overexpression of HIPK2 induces apoptosis in cultured sensory neurons.Conversely, targeted deletion of HIPK2 leads to increased expression of Brn3a, TrkA, and Bcl-xL, reduced apoptosis and increases in neuron numbers in the trigeminal ganglion.Together, these data indicate that HIPK2, through regulation of Brn3a-dependent gene expression, is a critical component in the transcriptional machinery that controls sensory neuron survival.

Affiliation: Department of Pathology, University of California San Francisco, San Francisco, CA 94143, USA.

ABSTRACTThe Pit1-Oct1-Unc86 domain (POU domain) transcription factor Brn3a controls sensory neuron survival by regulating the expression of Trk receptors and members of the Bcl-2 family. Loss of Brn3a leads to a dramatic increase in apoptosis and severe loss of neurons in sensory ganglia. Although recent evidence suggests that Brn3a-mediated transcription can be modified by additional cofactors, the exact mechanisms are not known. Here, we report that homeodomain interacting protein kinase 2 (HIPK2) is a pro-apoptotic transcriptional cofactor that suppresses Brn3a-mediated gene expression. HIPK2 interacts with Brn3a, promotes Brn3a binding to DNA, but suppresses Brn3a-dependent transcription of brn3a, trkA, and bcl-xL. Overexpression of HIPK2 induces apoptosis in cultured sensory neurons. Conversely, targeted deletion of HIPK2 leads to increased expression of Brn3a, TrkA, and Bcl-xL, reduced apoptosis and increases in neuron numbers in the trigeminal ganglion. Together, these data indicate that HIPK2, through regulation of Brn3a-dependent gene expression, is a critical component in the transcriptional machinery that controls sensory neuron survival.

fig2: HIPK2 suppresses Brn3a auto-regulatory activity and Brn3a downstream target genes. (A) Progressive down-regulation of TrkA and Bcl-xL, but not Bcl-2 or Bax, in trigeminal ganglion of Brn3a−/− mutants. The mRNA levels of TrkA, Bcl-2, Bcl-xL and Bax are determined in the trigeminal ganglion of wild-type and Brn3a−/− embryos from E12.5 to14.5 using qRT-PCR assays. Although the expression levels of these genes in Brn3a−/− are comparable to those in wild type at E12.5, mRNA levels of TrkA and Bcl-xL show significant down-regulation at E13.5. By E14.5, <20% of TrkA and Bcl-xL mRNAs are detected in the trigeminal ganglion of Brn3a−/−. In contrast, mRNA levels of Bcl-2 and Bax in Brn3a−/− are comparable to those in wild type at the same stages. Data represent mean ± SEM (n = 3). (B) Consistent with the qRT-PCR results, the amounts of TrkA and Bcl-xL protein show significant reductions in the trigeminal ganglion of Brn3a−/− at E15.5. In contrast, no reduction in Bax is detected. (C) HIPK2 inhibits Brn3a-mediated activation of luciferase construct Prox3, which contains synthetic DNA binding elements from brn3a enhancer. The luciferase activities of Prox3 are presented as mean ± SEM (n = 3, * indicates P < 0.01, t test). The amounts of DNA added to each reaction are indicated below each graph. All luciferase activities are normalized to Renilla luciferase reporter activity. (D) Consistent with its ability to suppress the luciferase activity of Prox3, expression of EGFP-HIPK2 in cultured trigeminal neurons using HSV leads to a progressive down-regulation of Brn3a mRNA. In contrast, expression of LacZ or EGFP in neurons has no effect on Brn3a mRNA level. Data represent mean ± SEM (n = 3, ns indicates no significant difference and * indicates P < 0.01, t test). (E) HIPK2 inhibits Brn3a-mediated activation of the TrkA luciferase activity. TrkA luciferase construct contains two Brn3a binding sites identified in the 5′-end of trkA promoter (Ma et al., 2003). TrkA luciferase activities, measured 24 h after transfection, are expressed as fold change compared with control. Values are expressed as mean ± SEM (n = 3). * Indicates P < 0.01 using t test. (F) HIPK2 down-regulates TrkA mRNA level to <10% of that in neurons expressing EGFP. In contrast, Brn3a up-regulates the mRNA of TrkA in cultured sensory neurons by at least 12-fold. Changes in the level of TrkA mRNA are determined using qRT-PCR assays and standardized to GAPDH. Neurons are collected for RNA extraction 48 h after infection. qRT-PCR assays are done in triplicates and at least three samples from each treatment are examined. Data represent mean ± SEM. (G) HIPK2 also inhibits Brn3a-mediated activation of Bcl-xL luciferase activity. Data represent mean 6 SEM (n = 3, * indicates P < 0.01, t test). (H) Expression of EGFP-HIPK2 down-regulates the endogenous level of Bcl-xL mRNA in cultured sensory neurons to <20% of that in neurons expressing EGFP. In contrast, expression of Brn3a increases Bcl-xL mRNA by more than threefold. Data represent mean ± SEM (n = 3).

Mentions:
The pro-survival effect of Brn3a depends on its auto-regulatory activity and the ability of Brn3a to up-regulate several prosurvival genes, including Trk receptors and members of the Bcl-2 family (Huang et al., 1999b; Smith et al., 2001; Ma et al., 2003; Trieu et al., 2003). Indeed, the mRNA levels of TrkA and Bcl-xL, as determined by quantitative RT-PCR (qRT-PCR) assays, showed progressive down-regulation in the trigeminal ganglia of Brn3a−/− mutants at E13.5 and E14.5, preceding the drastic increase in cell death at E15.5 to E17.5 (Fig. 2 A; Huang et al., 1999b). In contrast, expression of Bcl-2 and Bax in the trigeminal ganglion of Brn3a−/− mutants at the same stages remained unchanged (Fig. 2 A). Consistent with the qRT-PCR results, protein levels of TrkA and Bcl-xL showed significant reductions in the trigeminal ganglion of Brn3a−/− mutants at E15.5, whereas Bax remained unchanged (Fig. 2 B). To determine if the effect of HIPK2 on Brn3a-mediated gene expression was direct, we used luciferase constructs that contained three synthetic tandem repeats of Brn3a binding sites identified in the brn3a enhancer element (Prox3), which has been shown to be a reliable reporter for Brn3a activity. Consistent with previous data (Trieu et al., 1999), Brn3a activated the Prox3 reporter in a dose-dependent fashion, whereas HIPK2 by itself did not affect the baseline level of Prox3 luciferase activity (Fig. 2 C). Interestingly, addition of HIPK2 suppressed Brn3a-mediated activation of Prox3 (Fig. 2 C). To determine if expression of HIPK2 in sensory neurons leads to down-regulation of Brn3a in a more physiological setting, we used herpes virus vectors to introduce EGFP-HIPK2 into cultured sensory neurons (Coopersmith and Neve, 1999), achieving almost 100% infection efficiency at 50–100 multiplicity of infection (MOI). Subsequent to infection, total RNA was extracted and mRNA converted to cDNA. qRT-PCR assays were used to measure Brn3a mRNA levels, which were standardized to the endogenous GAPDH level. Our results indicated that, expression of EGFP-HIPK2 leads to a progressive down-regulation of Brn3a mRNA in sensory neurons. By 36 h after infection, Brn3a mRNA was reduced to <40% of that in control neurons infected with LacZ or EGFP virus (P < 0.01; Fig. 2 D).

fig2: HIPK2 suppresses Brn3a auto-regulatory activity and Brn3a downstream target genes. (A) Progressive down-regulation of TrkA and Bcl-xL, but not Bcl-2 or Bax, in trigeminal ganglion of Brn3a−/− mutants. The mRNA levels of TrkA, Bcl-2, Bcl-xL and Bax are determined in the trigeminal ganglion of wild-type and Brn3a−/− embryos from E12.5 to14.5 using qRT-PCR assays. Although the expression levels of these genes in Brn3a−/− are comparable to those in wild type at E12.5, mRNA levels of TrkA and Bcl-xL show significant down-regulation at E13.5. By E14.5, <20% of TrkA and Bcl-xL mRNAs are detected in the trigeminal ganglion of Brn3a−/−. In contrast, mRNA levels of Bcl-2 and Bax in Brn3a−/− are comparable to those in wild type at the same stages. Data represent mean ± SEM (n = 3). (B) Consistent with the qRT-PCR results, the amounts of TrkA and Bcl-xL protein show significant reductions in the trigeminal ganglion of Brn3a−/− at E15.5. In contrast, no reduction in Bax is detected. (C) HIPK2 inhibits Brn3a-mediated activation of luciferase construct Prox3, which contains synthetic DNA binding elements from brn3a enhancer. The luciferase activities of Prox3 are presented as mean ± SEM (n = 3, * indicates P < 0.01, t test). The amounts of DNA added to each reaction are indicated below each graph. All luciferase activities are normalized to Renilla luciferase reporter activity. (D) Consistent with its ability to suppress the luciferase activity of Prox3, expression of EGFP-HIPK2 in cultured trigeminal neurons using HSV leads to a progressive down-regulation of Brn3a mRNA. In contrast, expression of LacZ or EGFP in neurons has no effect on Brn3a mRNA level. Data represent mean ± SEM (n = 3, ns indicates no significant difference and * indicates P < 0.01, t test). (E) HIPK2 inhibits Brn3a-mediated activation of the TrkA luciferase activity. TrkA luciferase construct contains two Brn3a binding sites identified in the 5′-end of trkA promoter (Ma et al., 2003). TrkA luciferase activities, measured 24 h after transfection, are expressed as fold change compared with control. Values are expressed as mean ± SEM (n = 3). * Indicates P < 0.01 using t test. (F) HIPK2 down-regulates TrkA mRNA level to <10% of that in neurons expressing EGFP. In contrast, Brn3a up-regulates the mRNA of TrkA in cultured sensory neurons by at least 12-fold. Changes in the level of TrkA mRNA are determined using qRT-PCR assays and standardized to GAPDH. Neurons are collected for RNA extraction 48 h after infection. qRT-PCR assays are done in triplicates and at least three samples from each treatment are examined. Data represent mean ± SEM. (G) HIPK2 also inhibits Brn3a-mediated activation of Bcl-xL luciferase activity. Data represent mean 6 SEM (n = 3, * indicates P < 0.01, t test). (H) Expression of EGFP-HIPK2 down-regulates the endogenous level of Bcl-xL mRNA in cultured sensory neurons to <20% of that in neurons expressing EGFP. In contrast, expression of Brn3a increases Bcl-xL mRNA by more than threefold. Data represent mean ± SEM (n = 3).

Mentions:
The pro-survival effect of Brn3a depends on its auto-regulatory activity and the ability of Brn3a to up-regulate several prosurvival genes, including Trk receptors and members of the Bcl-2 family (Huang et al., 1999b; Smith et al., 2001; Ma et al., 2003; Trieu et al., 2003). Indeed, the mRNA levels of TrkA and Bcl-xL, as determined by quantitative RT-PCR (qRT-PCR) assays, showed progressive down-regulation in the trigeminal ganglia of Brn3a−/− mutants at E13.5 and E14.5, preceding the drastic increase in cell death at E15.5 to E17.5 (Fig. 2 A; Huang et al., 1999b). In contrast, expression of Bcl-2 and Bax in the trigeminal ganglion of Brn3a−/− mutants at the same stages remained unchanged (Fig. 2 A). Consistent with the qRT-PCR results, protein levels of TrkA and Bcl-xL showed significant reductions in the trigeminal ganglion of Brn3a−/− mutants at E15.5, whereas Bax remained unchanged (Fig. 2 B). To determine if the effect of HIPK2 on Brn3a-mediated gene expression was direct, we used luciferase constructs that contained three synthetic tandem repeats of Brn3a binding sites identified in the brn3a enhancer element (Prox3), which has been shown to be a reliable reporter for Brn3a activity. Consistent with previous data (Trieu et al., 1999), Brn3a activated the Prox3 reporter in a dose-dependent fashion, whereas HIPK2 by itself did not affect the baseline level of Prox3 luciferase activity (Fig. 2 C). Interestingly, addition of HIPK2 suppressed Brn3a-mediated activation of Prox3 (Fig. 2 C). To determine if expression of HIPK2 in sensory neurons leads to down-regulation of Brn3a in a more physiological setting, we used herpes virus vectors to introduce EGFP-HIPK2 into cultured sensory neurons (Coopersmith and Neve, 1999), achieving almost 100% infection efficiency at 50–100 multiplicity of infection (MOI). Subsequent to infection, total RNA was extracted and mRNA converted to cDNA. qRT-PCR assays were used to measure Brn3a mRNA levels, which were standardized to the endogenous GAPDH level. Our results indicated that, expression of EGFP-HIPK2 leads to a progressive down-regulation of Brn3a mRNA in sensory neurons. By 36 h after infection, Brn3a mRNA was reduced to <40% of that in control neurons infected with LacZ or EGFP virus (P < 0.01; Fig. 2 D).

Bottom Line:
Overexpression of HIPK2 induces apoptosis in cultured sensory neurons.Conversely, targeted deletion of HIPK2 leads to increased expression of Brn3a, TrkA, and Bcl-xL, reduced apoptosis and increases in neuron numbers in the trigeminal ganglion.Together, these data indicate that HIPK2, through regulation of Brn3a-dependent gene expression, is a critical component in the transcriptional machinery that controls sensory neuron survival.

Affiliation:
Department of Pathology, University of California San Francisco, San Francisco, CA 94143, USA.

ABSTRACTThe Pit1-Oct1-Unc86 domain (POU domain) transcription factor Brn3a controls sensory neuron survival by regulating the expression of Trk receptors and members of the Bcl-2 family. Loss of Brn3a leads to a dramatic increase in apoptosis and severe loss of neurons in sensory ganglia. Although recent evidence suggests that Brn3a-mediated transcription can be modified by additional cofactors, the exact mechanisms are not known. Here, we report that homeodomain interacting protein kinase 2 (HIPK2) is a pro-apoptotic transcriptional cofactor that suppresses Brn3a-mediated gene expression. HIPK2 interacts with Brn3a, promotes Brn3a binding to DNA, but suppresses Brn3a-dependent transcription of brn3a, trkA, and bcl-xL. Overexpression of HIPK2 induces apoptosis in cultured sensory neurons. Conversely, targeted deletion of HIPK2 leads to increased expression of Brn3a, TrkA, and Bcl-xL, reduced apoptosis and increases in neuron numbers in the trigeminal ganglion. Together, these data indicate that HIPK2, through regulation of Brn3a-dependent gene expression, is a critical component in the transcriptional machinery that controls sensory neuron survival.